Dipotassium adipate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/2013-09/2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP according to OECD guideline

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Principles of method if other than guideline:

/

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium adipate

EC Number:

242-838-1

EC Name:

Dipotassium adipate

Cas Number:

19147-16-1

Molecular formula:

C6H10O4.2K

IUPAC Name:

dipotassium adipate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Potassium adipate
- Physical state: white crystalline powder with lumps
- Lot/batch No.: 150413AK DRIED
- Expiration date of the lot/batch: 29/04/2014
- Stability under test conditions: not indicated
- Storage condition of test material:at room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: +/- 9 weeks old
- Weight at study initiation: variation was within +/- 20% of the sex mean
- Housing: in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet (e.g. ad libitum):Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum):Free access to tap water.
- Acclimation period:The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): 15 room air changes.hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark

Study design: in vivo (LLNA)

Vehicle:

other: Ethanol/water (7:3 v/v)

Concentration:

0%, 5%, 10%, 25% test substance (%w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility:25%, this was the highest concentration that could be prepared homogeneously
- Irritation: No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
TEST SUBSTANCE PREPARATION
Vehicle Ethanol/water (7:3 v/v) (ethanol: Merck, Darmstadt, Germany; water: Elix, Millipore S.A.S., Molsheim, France).
Rationale: Ethanol/water (7:3 v/v) was selected as suitable vehicle based on trial formulations performed at WIL Research Europe and test substance data supplied by the sponsor. Stability/solubility of the positive control in Ethanol/water (7:3 v/v) was not indicated.
Preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.

INDUCTION - DAYS 1, 2 AND 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The vehicle and positive control animals were treated in the same way as the experimental animals, except that the vehicle and/or positive control substance was administered instead of the test substance.

ECXISION OF THE NODES - DAY 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal)
with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING FOR RADIOACTIVITY - DAY 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4oC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS - DAY 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

/

Results and discussion

Positive control results:

The positive control group added to the study showed that the vehicle is suitable for eliciting an SI>3 (13.1) in this batch of animals and with the procedures used for this study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.3

Test group / Remarks:

3 groups of 5 females

Remarks on result:

other: The SI value calculated for the substance concentration of 25%,

Results 2

Key result

Parameter:

SI

Value:

ca. 0.9

Test group / Remarks:

3 groups of 5 females

Remarks on result:

other: The SI value calculated for the substance concentration of 10%,

Results 3

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

3 groups of 5 females

Remarks on result:

other: The SI value calculated for the substance concentration of 5%,

Results 4

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The DPM values for the substance concentration 0%, 5%, 10% and 25% were 468, 473, 430, 601 respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information

Conclusions:

Based on these results, POTASSIUM ADIPATE would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Executive summary:

Assessment of Contact Hypersensitivity to POTASSIUM ADIPATE in the Mouse (Local Lymph Node Assay).

The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2010),
EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay" EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test substance concentrations selected for the main study were based on the results of a pre-screen test.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 5%, 10% or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Ethanol/water) and another group of five animals received a positive control substance Alpha- Hexylcinnamaldehyde (HCA).

Three days after the last exposure, all animals were injected with3H-methylthymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of one ears of all animals at 25% (Days 1-3), which did not hamper scoring of the skin reactions.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5%, 10% and 25% were 473, 430 and 601 DPM respectively. The mean DPM/animal value for the vehicle control group was 468 DPM and a mean DPM/animal value of 6133 DPM was obtained from the positive control group.

The SI values calculated for the substance concentrations 5%, 10% and 25% were 1.0, 0.9 and 1.3 respectively.

Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 25%, POTASSIUM ADIPATE was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 25%.

The positive control group added to the study showed that the vehicle is suitable for eliciting an SI>3 (13.1) in this batch of animals and with the procedures used for this study.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Based on these results, POTASSIUM ADIPATE would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.